Abstract.From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (or subgroup of connexins) than to mouse Cx32 (B subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lungsimilar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two-to fourfold higher than in the corresponding adult tissues.Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (r = 580 ms at Vj of +50 mY). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into couplingdeficient human I-IeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.

Abstract. The coding sequence (333 amino acids) of a new connexin protein, designated mouse connexin37 (Cx37 or Cx37.6) due to the deduced theoretical molecular mass of 37.600 kD, has been determined from cDNA and genomic clones. As seen in other connexins, its gene has no introns within the coding region and the deduced amino acid sequence is predicted to have similar topology to other connexins that form intercellular channels. The amino acid sequence of mouse Cx37 is most similar to rat connexin43 (59% identity) and Xenopus connexin38 (66% identity) when compared from the NH2 terminus to the end of the fourth putative transmembrane region. When expressed in

When recombinant brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are purified by reverse-phase chromatography, these neurotrophins elute as two distinct peaks. This is also the case when naturally occurring BDNF is purified from brain tissue. As indicated by gelfiltration experiments, the peaks with shorter retention times correspond to neurotrophin dimers, those with longer retention times to monomers. In contrast, a BDNF mutant with a single aminoacid replacement (Arg-l+Lys) in the basic processing site common to all neurotrophin precursors elutes as a single peak. This peak is shown by gel-filtration chromatography to consist of dimers with a molecular mass almost twice that of wild-type dimers. N-terminal sequencing indicates an extension of 19 amino acids, including a glycosylated asparagine residue. The biological activity of the BDNF mutant ([R-1KIBDNF) is identical with that of wild-type BDNF when tested in a neuron survival assay. Using this assay, the biological activities of guanidine-hydrochloride-denatured neurotrophin monomers were found to be much lower than that of the dimers, and experiments with NT-3 monomers and NIH3T3 cells expressing trkC suggest that such monomers exist in solution in a conformation that prevents efficient interactions with neurotrophin receptors.Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4/5 are members of a gene family commonly referred to as the neurotrophins. All have been shown to prevent the death of a variety of cultured embryonic neurons (for a recent review, see Davies, 1994). The importance of NGF, BDNF and NT-3 for the normal development of the nervous system has also been demonstrated in a series of experiments involving the use of function-blocking antibodies (for NGF and NT-3), the targeted elimination of the neurotrophin genes (NGF, BDNF and NT-3) or of their respective receptor tyrosine kinase genes, trk, trkB and trkC, respectively. In all cases, substantial neuronal losses were observed in the peripheral nervous system (for reviews, see Barbacid, 1994;Davies, 1994).Mature, biologically active neurotrophins are cleaved from a pro-sequence at a common, basic consensus sequence of the furin type (Bresnahan et al., 1990) and they share a high degree of sequence similarity (-50%; for review, see Barde, 1990). They are basic, homodimeric, secretory proteins with a molecular mass of approximately 27 kDa (Bothwell and Shooter, 1977; Radziejewsh et al., 1992). Six strictly conserved cysteine residues form three intramolecular disulphide bridges in NGF and BDNF (Acklin et al., 1993) as a homodimer in solution, even at very low (0.1 pM) physiologically relevant concentrations (Bothwell and Shooter, 1977). The crystal structure of the NGF dimer revealed that it is stabilized by the apposition of large hydrophobic surfaces found in the protomers (McDonald et al., 1991). Many hydrophobic residues conserved in the neurotrophins contribute to the area of contact between the protomers (McDonald et ...

A primordial rhythm-generating neural network emerges during the segmental period of vertebrate hindbrain development, suggesting a common genetic basis to both the structure and network activity of the region. We show here that segmentation influenced a postsegmental developmental step by which a GABAergic rhythm generator was incorporated into the primordial network and increased rhythm frequency to near mature values. This process depended on specifications in r3 and r5 that controlled, on the basis of a two-segment repeat, later maturation of GABAergic inhibition.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are known to exist in solution as non-covalently linked homodimeric proteins. The recent elucidation of the crystal structure of the NGF homodimer, as well as the conservation of structural motifs in the neurotrophins, raised the possibility that neurotrophin heterodimers might also occur. The formation of BDNFNT-3 heterodimers was explored using a vaccinia virus expression system. Upon co-infection of cells with viruses expressing BDNF and NT-3, we could identify a BDNFNT-3 heterodimer as a biosynthetic product and separate it from the BDNF and NT-3 homodimers. We could also show that the BDNF/NT-3 heterodimers can be formed irrespective of wild-type or exchanged prosequences, indicating that prosequence specificity does not influence dimer formation. In all neuronal survival assays that were used, the BDNFNT-3 heterodimer was shown to be 10-fold less active compared with a mixture of BDNF and NT-3 homodimers. This lower specific activity was also measured in a neuronal population co-expressing receptors for BDNF and NT-3. The low biological activity of the heterodimer observed with neurons was not paralleled by a reduced ability of the heterodimer to interact with trkB or trkC receptors, as assessed by the induction of tyrosine phosphorylation of these receptors expressed by fibroblast cell lines.